The adrenal cortex of the guinea pig is composed of chromatically distinct outer and inner zones which can be separated by microdissection. In studies exploring the responsivity of the two zones to ACTH, the following observations have been made: 1) there is no steroidogenic response to ACTH by cells isolated from the inner zone; 2) cholesterol side-chain cleavage activity (rate-limiting in steroidogenesis) is significantly lower in the inner zone and is not modulated by ACTH, stress, or chronic dexamethasone suppression; 3) the content of cholesterol is 3-4 times higher in the outer zone. Thus, the guinea pig presents an interesting animal model to investigate steroidogenesis, cholesterol metabolism, and the mechanism of action of ACTH by performing experiments on the two adrenocortical zones in a parallel fashion. The regulation of adrenocortical steroidogenesis by ACTH is complex and only partially understood. The accepted obligatory steps include: stimulation of plasma membrane adenylate cyclase, increase in intracellular cAMP, and activation of cAMP-dependent protein kinase. The role of other kinases such as Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinase is less clear. A large number of proteins (membranous and soluble) which are phosphorylated in response to ACTH have been reported. To date, however, no regulatory phosphoprotein has been identified. Phosphoprotein phosphatases have not been examined. Based on the use of inhibitors of protein and RNA synthesis, a critical role for protein synthesis in the stimulation of adrenal steroidogenesis by ACTH has been proposed. Adrenal steroid production is rapidly activated and deactivated (approximately 2 min); such a process is considered too rapid to involve regulation at the level of translation. Regulation would, however, be compatible with protein modification. It is now well established that the covalent modification of protein is a crucial mechanism by which cellular processes are regulated. It is essential to identify and characterize the modified (eg. phosphorylated or dephosphorylated) steroidogenic regulatory protein. Such is a goal of the comparative approach using the guinea pig adrenal cortex model.